Method for rational mutagenesis of alpha/beta t-cell receptors and correspondingly mutated mdm2-protein specific alpha/beta t-cell ceceptores

ABSTRACT

The invention relates to the rational mutagenesis of polypeptides of α/β T-cell receptors that mediate an oncogen-specific T-cell response, nucleic acids encoding these and their use in the therapy, diagnosis and/or prevention of cancerous diseases. The invention further relates to a T-cell response-mediating MDM2-protein-specific α/β T-cell receptor, which has been rationally mutated by means of the method according to the present invention, and the uses thereof.

The invention relates to the rational mutagenesis of polypeptides of α/βT-cell receptors that mediate an oncogen-specific T-cell response,nucleic acids encoding these and their use in the therapy, diagnosisand/or prevention of cancerous diseases. The invention further relatesto a T-cell response-mediating MDM2-protein-specific α/β T-cellreceptor, which has been rationally mutated by means of the methodaccording to the present invention, and its uses.

The antigen recognition by T-lymphocytes (TLC) is critical for thegeneration and regulation of an effective immune response. Thecharacteristic T-cell line-marker is the T-cell-antigen-receptor (TCR).There are two types of TCR differentiated by sequence: the heterodimericα/β-TCR, and the structurally related γ/67 -TCR. The respective pairs ofchains are covalently linked by a disulfide bridge, and are associatedwith a set of five polypeptides, the CD3-complex, and together form theT-cell receptor-complex (TCR-CD3-complex). The α/β-TCR is thefunctionally most relevant, since it is expressed in more than 95% ofall T-cells, and mediates the primary immune response.

α/β-T-Cells can be separated in two different overlapping populations:One subgroup, which carries the CD4-marker and mainly supports theimmune response (T_(H)), and a subgroup which carries the CD8-marker andis essentially cytotoxic (T_(C)). CD8⁺-T-cellc recognize antigens inassociation with MHC-class-I-molecules. Such antigens, amongst others,can be tumor-specific or tumor-associated peptide antigens. Followingrecognition of the peptide antigens, the respective cell is killed inthat the T-cell lyses the target cell and/or induces apoptosis of thesetarget cells or releases cytokines (e.g. IL-2, IFN-γ). This constitutesan essential functional difference, compared to antibodies: TCRexclusively recognize peptide antigens in the context of theMHC-presentation, whereas antibodies recognize sequential orconformational peptide antigens independently from further accessorymolecules. Thus, TCRs are the suitable molecular tools in order torecognize tumor protein-derived antigens, and for coupling them directlyto a cytotoxic T-cell-response. In turn, antibodies primarily have thefunction of recognizing surface markers of cells as pathogenic, and tolabel them in order to be eliminated in the following by other effectorcells, e.g. macrophages, via phagocytosis.

Among the tumor associated peptide antigens (TAA) that are presented inthe context of MHC-class-I-molecules on the surface of tumor cells, theso-called “universal” TAA are of particular interest. These TAA aremainly derived from cellular proteins that are weakly expressed innormal cells and over-expressed in tumor cells. Belonging to theseproteins, amongst others, is the human homolog of the“mouse-double-minute-2” proto-oncogene (mdm2), the so-called “humanmdm2” or, abbreviated, “MDM2” proto-oncoprotein (Roth et al. 1998), thatis not only over-expressed in a variety of solid tumors but also inhematological neoplasia (malign hematological systemic diseases), AML,ALL and CLL (Zhou et al, 2000).

Oligopeptides of the MDM2-protein can be presented in the context withMHC-class-1-molecules on the cellular surface, and represent attractivetarget structures for CD8-positive T-cells. Here, the extent of thecourse of the T-cell response stays within a defined kinetic window(Kersh et al., 1998). The complex of peptide-MHC and TCR-CD3multimerises in order to effect an efficient signal transduction, inwhich the exact stoichiometry and the extent of the oligomerisation isstill controversial.

The present invention relates to a biochemical problem in the area ofapplied immunology & oncology: Focus of the invention is the developmentof highly-effective T-cells that are able to specifically recognize andlyse human (Hu) tumor cells via their cytotoxic effector-function. Forthis, CTL-clones which recognize specific TAA are isolated in atransgenic murine (Mu) or mouse-model (Stanislawski & Voss et al.,2001). Responsible for the oncoprotein derived peptide-recognition onthe side of the T-cells is the membrane stemming TCR that recognizes thecomplex out of membrane stemming MHC-molecule and presented peptide,which, on the side of the antigen presenting cell (APC) or also tumorcell, has arisen from the proteosomal processing of oncoproteins, andmediates an activating signal to the signal transduction cascade of thecytotoxic T-cell (CTL).

The prospective clinical use envisages isolating peripheric T-cells fromthe blood of a tumor patient, adding the gene of the TAA-specific TCR byadoptive gene technology based transfer, and, following massiveexpansion, re-infusing (Rosenberg, 1999; Schumacher, 2002).

The T-cell-receptor is a heterodimeric α/β-molecule, whose chains areeach uniquely spanning the transmembrane. Each chain consists out of twoglobular domains that have an immunoglobulin-like folding: the aminoterminal domain is designated as the variable domain (Vα or Vβ,respectively), since it is derived from genetic rearrangement, and isresponsible for the individual peptide recognition. The domain thatfollows is called the invariant or also constant domain (Cα or Cβ,respectively), since it is highly conserved, and essentially has aspacer function for the variable domain to the cellular membrane, aswell as regulatory proteins that bind to it. Finally, the section of atransmembrane region and a short carboxy terminal cytoplasmatic end,whereto the signal transducing CD3-complex is able to bind, do follow.This arrangement is valid to the same extent for human and for murineTCR: the foreign-species protein-backbone is superimposable with only amarginal difference of merely 1.04 Angstrom. In particular in theconstant domain numerous amino acids are homologous or identical,respectively (see FIG. 1), as exemplary shown for a murine (1) comparedto a human, HLA-A2.1-restringed TCR (1bd2). Although the T-cell-receptoris a heterodimer molecule made from two polypeptide-chains, in contrastto the antibody it has a monovalent binding site. Antibodies inprinciple are homodimers of heterodimeric subunits, from which abivalent antigen recognition of one and the same antibody results (i.e.monospecifc): two identical arms, each consisting of a heterodimericattachment of a heavy and a light chain, which therefore form anantigen-binding site, are covalently connected via a disulfide bridge.From this, contact areas result between the arms of an antibody that arenot present in a TCR: this generated the object (Atwell et al., 1997; WO96/27011) to modify the bivalent antigen recognition in such a way tolet each of both antigen-binding sites recognize a different antigen,i.e. to introduce a bispecificity into the complete antibody molecule.Finally, this exemplary provides a basis for an antibody that recognizesa tissue specific antigen as well as a pathogenic antigen. This goal cannot be realized in a TCR, since it structurally corresponds only to oneof both arms of an antibody, and is also monovalent, and thereforemandatory monospecific.

A different object results for the therapeutic use of TCRs: in that thepolyclonal T-cell population of the patient carries individual so-calledendogenic T-cell-receptors of unknown specificity. A functional TCR isformed out of the pairing of both chains via the endoplasmatic reticulum(ER)- and Golgi-processing pathway into a TCR/CD3-complex which isdirected to the cellular surface. Since the heterodimeric chains arefirst expressed separately and, in addition, a sufficient homologybetween the human and murine chains exists, the exogenically added TCRare able to pair with the endogenic chains, and elicit unknown, in theworst case unwanted, monospecificities (autoimmune reactions). In thesimplest case, the four chains as present (two endogenic and twoexogenic chains) in a T-cell being transduced with the genes for a TCRresult into four conceivable combinations, out of which two are unwantedHu α/Mu β and Mu α/Hu β-hybrids. Further, the situation gets morecomplicated, if several TCR-genes specific for different TAAs aretransduced, or due to the fact that in some instances in one T-cell, dueto an insufficient allelic exclusion of one of the two genomic α-chains,two functional TCRs are expressed. Furthermore, in the context of aclinical application, presumably non-clonal T-cell-populations will betransduced, such that out of this a multitude of conceivable hybrid TCRare present on the population level.

Such a hybrid form has not yet been shown, nevertheless, based on thestructural data, such as TCR-protein crystal structures present so far(Garcia et al., 1998; Ding et al., 1998), it can not be excluded.Nevertheless, it could be shown in the laboratory of the inventors thata foreign single mouse-TCRβ-chain that is introduced into human T-cellsis only expressed on the surface if it is able to pair with theendogenic human TCRα-chains. Since this could be shown, and the exogenicexpression was amplifiable in case of partially humanized β-chains orwas diminishable through the introduction of point mutations thatinterfere with the pairing (see approach “compensated introduction of anexposed carrier of charge” described below), this is a strong indicationtowards the presence of hybrid TCRs. It is therefore mandatory for anincreased safety in the use of TCR to exclude such hybrid forms as muchas possible.

One resolving way for an avoidance of the unwanted pairing of chains isthe design of single chain T-cell receptors. This approach was initiallydeveloped for antibodies (Eshhar et al., 1993; 2001), but could betransferred to the latter based on the structural homologies betweenantibodies and T-cell receptors (Chung et al., 1994; Weijtens et al.,1998; Willemsen et al., 2000). For this, the variable domains arecovalently linked one with the other via a short peptide, a “linker”,omitting one of the two constant domains. Such constructs can be freelydesigned by genetic engineering and guarantee for a biochemicallycoupled 1:1-stoichiometry of the heterodimeric variable domains (forthis, see also FIG. 3).

Alternatively, short peptide sequences are genetically attached to therespective chains of the heterodimeric molecule that function asaffinity-tags that provide for a specific pairing of chains: for this,carboxy-terminal “tags” of 30 amino acids in length, so-called “leucinezipper”, were added to T-cell receptors as a dimerisation motif (Changet al., 1994). The latter methods have the drawback that they due totheir recombinant character represent potential foreign antigens whichlead to rejecting reactions in the acceptor organism.

In addition, the modified primary- and tertiary structure being markedlydifferent from the wild type structure of human as well as murine TCRimpede the stability and functionality of these chimeric constructs.

It is therefore the object of the present invention to provide a methodthat allows for a production of recombinant TCRs in such a way thatpreferably the externally introduced TCR-chains are pairing, and do notform mixed pairs with the endogenic chains of the T-cell, without at thesame time affecting their functionality and stability.

According to the invention, this object is solved by a method forproducing a heterodimeric specific wild type- or chimeric T-cellreceptor (TCR) containing a first chain and a second chain that interactone with another at least at one surface, wherein the at least onesurface is subjected to a rational mutagenesis, such that the at leastone surface of the first chain or the surface of the second chaincomprises a sterically projecting group, that interacts with asterically recessed group on the at least one surface of thecorresponding first chain or second chain. Preferably, the stericallyprojecting group that is comprised by the at least one surface of thefirst chain or the surface of the second chain is a charged and/or polargroup, and further preferred, the sterically recessing group on the atleast one surface of the corresponding first chain or second chain is anopposingly charged and/or polar group. Here, it can be sufficient tointroduce an exposed charge on one side, if the opposing contact areacan cooperatively compensate for this modification, or it can berequired to complementary introduce an opposing recessed charge. In thefollowing, this novel approach with TCR shall be designated anddescribed as the “compensated introduction of an exposed carrier ofcharge” (FIG. 11). Each “+”—but also “−”—symbol indicates a true chargeor a polar charge. A true charge (e.g. the charged guanidium-group ofarginine) can only be sufficiently compensated by several polar groups(e.g. carbonyles of the peptide groups) of several surfaces (aminoacids). This is depicted by the non-stoichiometric illustration of thecharge symbols: in many cases a sterically exposed true charge of theone chain (defined as sterically projecting group) projects into arecess of the cavity of the other chain (defined as sterically recessedgroup), which is lined with several polar or also charged groups. Eventrue charges are not directly compensated 1:1, since this also dependsfrom the respective (dielectric) availability and distance ofneighboring opposing charges. In view of the sterics as well as thecharge, the respective groups need not to be directly converted 1:1, butthis requires an individual structural analysis, optionally, e.g. byomission of a polar group within the cavity containing the chargecarrier, in order to achieve an as much as possible compensatory effect.Not only the state V, wherein a respective inversely related chargecarrier-surface is missing on the respective chains, corresponds to thewild type, but each of the states I-IV can also correspond to theinitial status of the polypeptide chains as found.

Here, the possibility exists to exclusively invert the given stericrelations (I/II or III/IV in FIG. 11, respectively), to invert thecharges (I/IV or II/III in FIG. 11, respectively) or both (II/IV orI/III in FIG. 11, respectively). The particular case of an inversion ofthe charged and/or polar sterics (I/II of the FIG. 11) functions asexample being experimentally shown on T-cell receptors (FIG. 2).

The method according to the invention comprises the steps of (a)providing the DNA-molecules, comprising the coding regions for the atleast one surface to be mutated of the first chain or second chain in ajoint or separate mutagenesis-vector system(s), (b) mutagenesis of theDNA-molecules in a manner known as such, wherein the nucleic acidsequence encoding for the at least one surface is modified compared tothe initial sequence in such a way that, in the at least one surface ofthe first chain or the at least one surface of the second chain, asterically projecting group, preferably charged and/or polar group isintroduced, and in the corresponding interacting at least one surface ofthe second chain or the first chain, a sterically recessed group,preferably reversely charged and/or polar group, can be introduced,whereby individual mutated fragments are produced, c) translation of atleast two of the individual mutated fragments from step b), such thatthe pairing of the heterodimeric specific first-chain/second-chain TCRbeing mutated at least one surface is selectively promoted, and d)presentation of the heterodimeric first-chain/second-chain TCR by aT-cell.

In the context of the present invention by a “surface” the area of achain of a TCR shall be understood which interacts with a particulararea of the second chain of the TCR. This interaction, either alone orin connection with others, leads to the formation of pairs of chainswhich form the active TCR. The sum of the interactions is based onelectrostatic, dipole-dipole-, Van der Waals-contacts, and hydrophobicinteractions that are determined by the amino acid sequences, as well asthe structural positioning of the polypeptide chains amongst each otherand to each other. In case of a punctual change, the secondary-,tertiary- and quartery structure of the heterodimer should beunmodified. Without wanting to be bound to a particular mechanism ofaction, the inventors are assuming that generally the method of thepresent invention is aiming at influencing the combination of charge andsterics of side chain(s) without modifying the protein backbone. Themodification of steric situations between interacting surfaces isinsufficient in many cases, since even sterically large side chains(valine, phenylalanine) do modify the local structure by replacment ofneighboring side chains, whether in relation to the own chain (surface)or the complementary chain (surface), in such a way that these aresterically accommodated within the contact area, without an effect onthe pairing of the chains (surfaces). An effective contribution isperformed by the rejection based on charge differences, since punctualcharges, according to the law of Coulomb, function spherically in spacewith 1/Dr². The affecting force exponentially decreases with anincreasing distance and thus provides for a locally restricted effect ofthe punctual charge, without a far-reaching effect on the tertiary andquartery structures. The dielectric constant D is lower in apolarmilieus, as is the case in the inside of proteins or also at contactareas of subunits, and therefore the force that is acting on opposingcharges is stronger. In order to amplify the effect on the surfaceinteractions that is potentially induced (or is missing) by stericrejection (or even to initiate it), amino acid residues are chosen thathave sterically exposed side chains with charges (e.g. arginine, lysine,glutamate), pH-inducible charges (e.g. histidine) or polar groups (e.g.glutamine). Preferably, this takes place by a modification on the levelof the primary structure of the chain, that is by amino acid exchangesinside the same. The punctual effect of the sterics as well as thecharges of the two interacting surfaces (e.g. two interacting aminoacids of opposing charge and reciprocal sterics) guarantees for aself-integrity of the sterical structures of the affected chains, but,nevertheless, in case of the interaction of two exposed or recessedidentically charged surfaces, as would be the case for the interactionof an unmodified with a mutated chain, by the sterically limiteddistortion, induces a weakening of the pairing, whereas thecomplementary surfaces are neutralizing each other with respect tosterics and charge.

An alternative of the method according to the invention relates to amethod, wherein the above-mentioned step c) above is replaced by thefollowing steps: (c′), optionally, sub-cloning of the mutated fragmentsinto suitable transfection-vector systems or virus-derived transductionsystems, (c″) transfection or co-transfection or transduction of atleast two of the mutated fragments into a mutant TCR-deficient T-cell,and (c′″) expression of the heterodimeric first-chain/second-chain TCRin a recombinant T-cell. This alternative relates to the transfer of thegenetic construct and its subsequent expression directly into arecombinant T-cell. Whilst this alternative is preferred, in anadditional alternative of the method according to the invention, step c)above can be replaced by the following steps: c′) In vitro-translationor in vivo-translation of at least two of the individual mutantfragments from step b) and, optionally, subsequent isolation and/orpurification of the translated mutant fragments, such that the pairingof the heterodimeric specific first-chain/second-chain TCR being mutatedon at least one surface is selectively promoted, and c″) introduction ofthe mutated specific first-chain/second-chain TCR into a T-cell.

For this, an expression of the mutated TCR occurs outside of the finallyanticipated presenting T-cell, and a subsequent introduction of the TCRinto the same. In case of the in vitro translation, the translation canoccur in cell-free systems which are commercially available. The“translation”, nevertheless, also comprises the purely syntheticproduction of the peptide chains as is explained in more detail furtherbelow in connection with the peptides. In case of the in vivotranslation, this can take place in a suitable host cell that has beentransformed with an expression construct of the chain in advance, andsubsequently produces it. Suitable vectors and methods for expressionare sufficiently known to the person of skill in the art. Following theexpression, it can be required to either purify the expression productsfrom the cells or to extract them from the medium, into which they haveoptionally been excreted by the host cell. Suitable host cells are alsoknown and can be yeast, CHO-cells, insect cells, bacteria or other.

The introduction into the T-target cells can take place using any knownmanner that allows for a subsequent presentation of the TCR by theT-cell. Strategies are, for example, by means of the induction ofphagocytosis by the cells or a method wherein the introduction occurs bylipid-mediated transfer, such as via micelles or liposome transfer. Anoverview about the use of liposomes, amongst others, is provided by thearticle of Banerjee R. Liposomes: applications in medicine. J BiomaterAppl 2001 July;16(1):3-21. The transfer via micelles is known to theperson of skill in the art from numerous publications.

Preferably, according to the invention, as heterodimeric specific wildtype or chimeric T-cell receptor (TCR), an alpha/beta TCR, gamma/deltaTCR, a humanized or partially humanized TCR, a TCR being provided withadditional (functional) domains, a TCR being provided with alternativedomains, e.g., a TCR being provided with a different transmembranedomain as membrane anchor, is modified.

Backstrom et al. (Backstrom B T, Hausmann B T, Palmer E. Signalingefficiency of the T cell receptor controlled by a single amino acid inthe beta chain constant region. J Exp Med. 1997 Dec. 1;186(11):1933-8)describe a Gln₁₃₆Phe-mutation in the beta-TCR. The mutation is localizedin the “beta chain connecting peptide domain”, next to thetransmembrane. The exchange as described is positioned far away from thepoint mutations that have been determined as useable for the rationalmutagenesis by the present invention. In addition, an effect on thepairing of the chains is not analyzed, but exclusively a functionalityof the TCR.

Backstrom et al. (Backstrom B T, Milia E, Peter A, Jaureguiberry B,Baldari C T, Palmer E. A motif within the T cell receptor alpha chainconstant region connecting peptide domain controls antigenresponsiveness. Immunity. 1996 November;5(5):437-47.) describe chimericTCRs, whose points of fusion are positioned beyond the terminal intrachain-cysteines, and, in particular, are related to a motif of the“alpha chain connecting peptide domain”, FETD×NLN. Both regions ofmutations are positioned closely to the transmembrane domain, i.e. faraway from the point mutations being described here as essential.Reciprocal amino acid exchanges (knob-hole) are not made.

Li et al. (Li Z G, Wu W P, Manolios N. Structural mutations in theconstant region of the T-cell antigen receptor (TCR) beta chain andtheir effect on TCR alpha and beta chain interaction. Immunology. 1996August;88(4):524-30 and WO 97/47644 and WO 96/22306 describe theanalysis of pairing of TCRs. The pairing is analyzed by immuneprecipitation and 2D-gel electrophoresis with a previous metaboliclabeling of the chains. The authors created chimeric betaTCR to amolecule being immunologically irrelevant, and determined, whether thesechimera, having different lengths but still corresponding to the wildtype of the beta-chain, paired with the alpha-chain. The chimerascomprise different regions of, in particular, the constant domain, butdo not indicate the point mutations that are described in the context ofthe present invention. The pairing-properties are neither examined underthe effect of the mutagenesis on both chains nor in the context of asteric inversion (knob-hole).

The authors, nevertheless, draw two conclusions that are very importantfor the invention: On the one hand, the constant domain is criticallyresponsible for the pairing, whereat, in particular, the regionSer₁₈₈-Leu₂₁₃ of betaTCR which particularly contains many basic aminoacids (arginine₂₀₈ or arginine₁₉₅ of the 1, respectively), and is likelyto be essential for the pairing, would be of interest. Nevertheless, thepublication does not elaborate on this.

It was already attempted to resolve the problem of the specificmanipulation of the interaction of the molecule by stericallycomplementary groups (Belshaw et al., Angew. Chem. Int. Ed. Engl. 34(1995), 2129-2132); and this was exemplified based on antibodies (WO96/27011; Atwell et al., 1997; Carter, 2001): for this, the heavy chainsof two different antibodies of a different epitope-specificity shouldspecifically pair one with the other, by sterically inverting contactingamino acid side chains at the contact position of both chains in theirsteric distribution in space: one small amino acid being present in thewild type interacting with an amino acid with a large side chain of theother chain is genetically mutated to an amino acid with a large sidechain, whereas the large amino acid partner is exchanged into a smallresidue (English: “knob-hole”-model). In case of the pairing of chainsof both mutated chains, again a small and a large amino acid again domeet each other, nevertheless with a steric inversion. If the directperiphery of the point mutants should allow for this steric exchange,the actual functionality of the heterodimeric molecule (that is, theepitope-recognition) should not be affected. WO 96/27011 describesbispecific antibodies, immune adhesines or chimeras thereof. Thisapproach, nevertheless, completely ignores the additional control of theinteraction of surfaces by the introduction of charge differences, andtherefore is also only a 1-parametric concept. In the approach asdescribed herein, a strict combinatorial 2-parametric concept ispresented which takes into account both the sterics as well as thecharge, and therefore implies that a 1-parametric approach in manyobjects does not comparably effectively manipulate the chain-pairing inthe desired manner. The “knob hole”-model and the model “compensatoryintroduction of exposed charge carriers” being described herein thus areto be regarded as independent strategies. In addition, the latter modelgives significantly more opportunities regarding the choice of themutations to be introduced, and therefore a broader spectrum of possiblesolutions.

Atwell et al. seeked to produce bispecific antibodies by linking the twohalves of epitope-different antibodies one with the other at theircontact positions (CH₃-domain) of the heavy chains (by “knob-hole”). Indoing so, there are essential differences compared to the presentinvention which are to be taken into account for a transfer of theprinciple from antibodies to TCRs. In contrast to the monovalent TCR, anantibody is bivalent. The result is a structurally heterodimericantibody that recognizes two different epitopes (bispecific). Theapproach according to the invention takes another direction: here, it istried to selectively link the two predestinated chains of a monospecificTCR, in order to not generate functionally heterodimeric TCRs havingpotentially monospecificities that divert from the monospecificity asdesired.

The selection of the amino acids to be exchanged occurred according tothe invention by “rational design”: for this existing crystal structuresof TCRs were studied by means of structure-representing software, andamino acid candidates were determined for punctual mutagenesis. Thequality of the amino acid exchanges (anticipating the set of 20codogenic natural amino acids in mammals) was each individually assessedaccording to steric circumstances as present in interacting amino acidpairs, respectively, by taking into account the surrounding of directlyneighboring amino acids. The model system that was established in orderto study the effect of the point mutants is described in more detailbelow.

Therefore, the method for rational mutagenesis of TCR according to theinvention is neither disclosed nor proposed by the above mentionedpublications as well as in the residual literature. The point mutationsin each chain that have been introduced according to the invention shalllead to the fact that preferentially the externally introduced TCRchains are pairing, and that no mixed pairs are formed with theendogenic chains of the T-cells. This is an essential contributionregarding the issue specificity and therefore safety of the T-cellresponse.

It has to be noted that, in contrast to the state of the art, pointmutants are markedly lower immunogenic than introduced affinity-tags or“linkers” comprising several amino acids, such as provided by, e.g., thesingle chain-TCR concept. In addition, the point mutants are nearlyidentical to the wild type chains that, until now, do have the strongestfunctional effectivity. All TAA-specific TCR that have been developedand will be developed, whether of murine or possibly also human origin,which shall be used in future in the adoptive immunotherapy by genetransfer into human T-cells of tumor patients, can easily be providedincluding these mutations. Therefore, the presented approach could finda wide-spread use in the clinical application (Bolhuis et al., 1998;Cavazzana-Calvo et al., 2000).

In a variant of the method according to the invention the amino acidsthat are first introduced after the mutagenesis of the DNA-molecules arefurther suitably chemically modified. Additional mutations can also beintroduced non-chemically, such as, for example, by genetically producedpoint mutations from “phage display”, in order to thereby introduce asterically projecting group or a sterically recessing group. This meansthat first an amino acid is introduced which functions as the initialbasis for the projecting group that is finally present. Suitablemodifications therefore are amino acid derivates that are modified bychemical means, such as, for example, methylation (e.g. α-methylvaline),amidation, in particular of the C-terminal amino acid using an alkylamine (e.g. ethylamine, ethanolamine, and ethylendiamine), andmodifications of an amino acid side chain, such as, for example,acylation of the ε-amino group of lysine. Other amino acids that can beincorporated into the chain include any of the D-amino acids whichcorrespond to the 20 L-amino acids that are commonly found in proteins,or iminio amino acids, rare amino acids, such as, for examplehydroxylysine, or non-protein amino acids, such as, for examplehomoserine and ornithine. A modified chain can exhibit one or several ofthese derivates, and D-amino acids. The chain can be synthesized by achemical method, in particular using an automated peptide synthesizer orcan be produced by a recombinant method. Modifications of the C-terminusinclude esterification and lactone formation. N-terminal modificationsinclude acetylation, acylation, alkylation, pegylation, myristylation,and the like.

Nevertheless, usually the amino acids that are introduced after themutagenesis of the DNA-molecules with the method according to theinvention will directly provide the sterically projecting, preferablycharged and/or polar groups or the sterically recessing, preferablyconversely charged and/or polar groups, without that a furthermodification is required. A particularly preferred mutagenesis methodaccording to the invention leads to an exchange of the amino acids ofthe first by those of the second chain or vice versa, wherein the aminoacids that are introduced by the mutagenesis of the DNA-molecules areselected in such a way that a reciprocal exchange of the amino acids atthe surfaces of the interacting chains des TCR is achieved.

In the context of the present invention, by sterically recessing,preferably charged and/or polar group, any chemically group beingattached to each of the chains to be mutated shall be understood whichsterically occupies less space compared to its corresponding earlierpresent group, and preferably carries a full or the fraction of a fullcharge unit. A charge is introduced which is complementary to thesterically projecting charged and/or polar group, either in the recessedgroup itself, or the net charge of the cage surrounding the stericallyprojecting charged and/or polar group is reverted by introducing acharge, or by the removal of a charge in the respective group (FIG. 11).Thus, the amino acid that is introduced after the mutagenesis of theDNA-molecules which introduces a sterically recessing group compared tothe initial sequence can be selected from glycine, serine, threonine,alanine, without being limited thereto. Here, serine and threonine arethose recessed groups that by themselves carry a partial charge. The useof glycine and alanine implies the emphasis of the remaining net chargeof the surrounding cage (e.g. partial charges of the peptide bonds inthe protein-backbone). Similarly, in the context of the presentinvention, as sterically processing group any chemically group beingattached to each of the chains to be mutated shall be understood whichsterically occupies more space compared to its corresponding earlierpresent group, and preferably carries a full or the fraction of a fullcharge unit. Thus, the amino acid that is introduced after themutagenesis of the DNA-molecules that introduces a sterically projectinggroup compared to the initial sequence can be selected from lysine,arginine, histidine, cysteine, glutamine, glutamate and tyrosine,without being limited thereto.

In another additional aspect of the present invention, in a methodaccording to the invention at least two surfaces of a TCR-chain aresimultaneously subjected to mutagenesis. Thereby, possibly a furthercontrolled pairing characteristic of the corresponding TCR can beachieved by means of the accumulation of several mutations, such as, forexample, an energetically stronger or less strong pairing kinetics,compared to the non-modified state. A method for optimizinginterchanging effects of several surfaces is represented by the“phage-display” method.

According to a further method according to the invention, the surfacesthat are accordingly involved in interchanging effects can be localizedin the variable domains of the TCR-chains. The surfaces that areaccordingly involved in interchanging effects can, nevertheless, also belocalized in the constant domains of the TCR-chains. The localization ofthese modifications is, amongst others, dependent from the desiredspecification of the respective TCR and its specific interactions.

Thus, according to a particular method according to the invention, thedomains of the TCR-chains to be mutated can be selected from mammalian,and there, in particular, from human and/or mouse domains. Thereby,particularly preferred is a mutagenesis according to the presentinvention, wherein the rational mutagenesis of the TCR-chainssimultaneously leads to a humanization of the TCR. Therefore, thismutagenesis, amongst others, leads to an improved compatibility of theTCR.

Therefore, the present invention essentially provides the threecomponents of:

-   -   a) The approach being independent from the “knob hole”-model, to        introduce charge into the inside of the complementary chain by        means of large steric groups and by small steric groups        themselves or by the omission of charged groups, in order to        arrive at a charge compensation of the introduced charge. This        is a strictly 2-parametric approach. Here, for the first time,        it was attempted to apply this model to the contact area between        the heterodimeric chains of a monovalent TCR, whereas so far in        antibodies these were referred to the contact area between the        heavy chains of a bivalently structured antibody.    -   b) The experimentally novel of the present invention is the        fact, that a pair of amino acids in the TCR, for which the        described model is applicable (αTCR/Gly¹⁹² and βTCR/Arg²⁰⁸ of        the MDM2(81-88)-specific TCR, FIG. 5) is determined, and the        quality of the exchanges (^(Arg)208^(Gly) and ^(Gly)192^(Arg))        is defined. This is about a 1:1 inversion of the steric and        charge-dependent situations as found, and corresponds to the        transition from status I (wild type) to status II (1        mutation/chain) of FIG. 11 or 2, respectively: The introduced        positive charge of the arginine is compensated by the residual        carbonyles of the polypeptide fiber of the complementary chain.        An additional indicator for a charge neutralization being        comparable to the wild type is the modeling capacity being        supported by the protein structure-database of hydrogen bridges        between the guanidinium-group of the arginine and the carbonyles        of the main chain both for status I as well as for II for the        crystal structure of an also murine TCR (1.pdb of FIG. 6).    -   c) The pair of amino acids to be exemplary determined here        should be chosen in such a manner that said pair can be        generalized from one to all murine TCRs, in order to also        provide future murine TCRs being specific for other TAAs with        these mutations, and, on the other hand, can also be extended to        human TCRs. The constant domain was chosen, since here a        sufficient homology between human and murine TCR exists, in        order to assume that the selected mutations do have a comparably        detrimental effect for hybrid Hu/Mu TCR, as was exemplary shown        for murine “hybrid” TCR in the present invention.

It has to be taken into account that other amino acid exchanges at therespective position can also have a comparable effect. Furthermore, anoptimization through additional cumulating exchanges in the surroundingof the respective point mutations is conceivable.

A further aspect of the present invention relates to a method whereinthe alpha- and beta-chains of a MDM2(81-88)-specific TCR are used as thealpha- and beta-chains, and wherein the Gly₁₉₂ of the constant region ofthe alpha-chain and the Arg208 of the constant region of the beta-chainare exchanged by Arg₁₉₂ in the constant region of the alpha-chain and byGly₂₀₈ in the constant region of the beta-chain. Based on this TCR, forthe first time the principle according to the invention couldsuccessfully be applied.

Further preferred is a method according to the invention wherein aretroviral vector, in particular pBullet, is used as transfectionsystem. In addition, IRES-elements can be used in said vectors.

A further aspect of the present invention relates to a mutated first(alpha-) or second (beta-) chain of a TCR that is produced according toa method according to the present invention. Further particularlypreferred is a TCR that is mutated according to the invention, inparticular a mutated MDM2(81-88)-specific TCR, wherein said TCR exhibitsat least one mutated alpha- and beta-chain. This mutated TCR accordingto the present invention can also be present in form of a fusionprotein, comprising the polypeptides modified according to the inventionor parts thereof. The fusion protein can be characterized in that itcomprises the ζ-region of CD3 or CD8 or CD16 or parts thereof, inparticular the ζ-region von human CD3 or CD8 or CD16 or parts thereof.In particular, the fusion protein according to the invention cancomprise a fusion protein of the 1-chain of the CD3-complex orITAM-motifs of the ζ-chain or parts thereof, in particular the ζ-chainof human CD3 or parts thereof. The fusion protein can furthermore becharacterized in that it comprises CD8α or the Lck-bindiung motif ofCD8α or parts thereof, in particular of human CD8α.

A further aspect of the present invention relates to an isolated nucleicacid comprising a sequence encoding for a mutated first (e.g. alpha-) orsecond (e.g. beta-) chain of a TCR according to the invention. Thisnucleic acid according to the invention can be a DNA, RNA, PNA (peptidenucleic acid) or p-NA (pyranosyl nucleic acid), preferably a DNA, inparticular a double-stranded DNA having a length of at least 8nucleotides, preferably with at least 18 nucleotides, in particular withat least 24 nucleotides. The nucleic acid can be characterized in thatthe sequence of said nucleic acid exhibits at least one intron and/or apolyA-sequence. It can also be present in form of its antisensesequence.

A further aspect of the present invention relates to a DNA- orRNA-vector molecule which comprises at least one or several nucleicacid(s) according to the invention and which can be expressed in cells.For the expression of the respective gene, in general a double-strandedDNA is preferred, whereby the DNA-region encoding for the polypeptidesis particularly preferred. This region starts with the first start-codon(ATG) being positioned in a Kozak consensus sequence (Kozak, 1987,Nucleic. Acids Res. 15:8125-48) up to the next stop-codon (TAG, TGA orTAA, respectively) that is positioned in the same reading frame to theATG. A further use of the nucleic acid sequences according to theinvention is the construction of anti-sense oligonucleotides (Zheng andKemeny, 1995, Clin. Exp. Immunol. 100:380-2) and/or ribozymes(Amarzguioui, et al. 1998, Cell. Mol. Life Sci. 54:1175-202; Vaish, etal., 1998, Nucleic Acids Res. 26:5237-42; Persidis, 1997, Nat.Biotechnol. 15:921-2). Using anti-sense oligonucleotides, one can reducethe stability of the nucleic according to the invention and/or inhibitthe translation of the nucleic according to the invention. Thus, forexample, the expression of the respective gene in cells can be reducedin vivo as well as in vitro. Therefore, oligonucleotides can be suitableas therapeutics. This strategy is, for example, suitable also for skin,epidermal and dermal cells, in particular, if the antisenseoligonucleotides are complexed with liposomes (Smyth et al., 1997, J.Invest. Dermatol. 108:523-6; White et al., 1999, J. Invest. Dermatol.112:699-705). A single-stranded DNA or RNA is preferred for use as aprobe or as “antisense” oligonucleotide.

In addition to the natural nucleic acids that have been isolated fromcells, all nucleic acids according to the invention or their parts canalso be produced synthetically. Furthermore, in order to work theinvention, a nucleic acid can be used that has been syntheticallyproduced. Thus, for example, the nucleic acid according to the inventioncan be chemically synthesized based on the protein sequences asdescribed by reference to the genetic code e.g. according to thephosphotriester-method (see, e.g. Uhlmann, E. & Peyman, A. (1990)Chemical Reviews, 90, 543-584).

In general, oligonucleotides are rapidly degraded by endo- orexonucleases, in particular by DNases and RNases that are present incells. It is therefore advantageous to modify the nucleic acid in orderto stabilize them against the degradation, such that a highconcentration of the nucleic acid in the cell is maintained over a longtime period (WO 95/11910; Macadam et al., 1998, WO 98/37240; Reese etal., 1997, WO 97/29116). Typically, such stabilization can be achievedby introducing one or several internucleotide-phosphorous groups or byintroducing one or several non-phosphorous-internucleotides.

Suitably modified internucleotides are summarized in Uhlmann and Peymann(1990 Chem. Rev. 90, 544) (WO 95/11910; Macadam et al., 1998, WO98/37240; Reese et al., 1997, WO 97/29116). Modifiedinternucleotide-phosphate residues and/or non-phosphorous bridges in anucleic acid that can be employed in a use according to the invention,for example, contain methyl phosphonate, phosphorothioate,phosphoramidate, phosphorodithioate, phophatester, whilstnon-phosphor-internucleotide-analogs, for example, contain siloxanebridges, carbonate bridges, carboxymethylester, acetamidate bridges,and/or thioether bridges. It is further intended that thesemodifications improve the shelf-life of a pharmaceutical compositionwhich can be employed in a use according to the invention.

A further aspect of the present invention relates to a vector,preferably in form of a plasmid, shuttle vector, phagemid, cosmid,expression vector, adenoviral vector, retroviral vector (Miller, et al.“Improved retroviral vectors for gene transfer and expression”,BioTechniques Vol. 7, No. 9, p 980, 1989) and/or gene-therapeuticallyeffective vector containing a nucleic acid according to the invention.

Thus, the nucleic acid according to the invention can be contained in avector, preferably in an expression vector or gene-therapeuticallyeffective vector. Preferably, said gene-therapeutically effective vectorcontains T-cell specific regulatory sequences that are operativelylinked with the nucleic acid according to the invention. The expressionvectors can be prokaryotic or eukaryotic expression vectors. Examplesfor prokaryotic expression vectors are e.g. the vectors pGEM orpUC-derivates for the expression in E. coli and for eukaryoticexpression vectors e.g. the vectors p426Met25 or p426GAL1 (Mumberg etal. (1994) Nucl. Acids Res., 22, 5767-5768) for the expression inSaccharomyces cerevisiae, e.g. Baculovirus-vector, such as disclosed inEP-B1-0 127 839 or EP-B1-0 549 721 for the expression in insect cells,and e.g. the vectors Rc/CMV, and Rc/RSV or SV40-vectors that arecommonly available for the expression in mammalian cells.

In general, the expression vectors do also contain promoters that aresuitable for the respective host cell such as, for example, thetrp-promoter for the expression in E. coli (see, e.g., EP-B1-0 154 133),the Met 25, GAL 1 or ADH2-promoter for the expression in yeasts (Russelet al. (1983), J. Biol. Chem. 258, 2674-2682; Mumberg, supra), thebaculovirus-polyhedrin-promoter for the expression in insect cells (see,e.g., 13. EP-B1-0 127 839). For the expression in mammalian cells, forexample, promoters are suitable that allow for a constitutive,controllable tissue specific, cell cycle specific or metabolicallyspecific expression in eukaryotic cells. Controllable elements accordingto the present invention are promoters, activator sequences, enhancers,silencers and/or repressor sequences. Examples for suitable controllableelements that allow for the constitutive expression in eukaryotes arepromoters that are recognized by the RNA polymerase III or viralpromoters, CMV-enhancers, CMV-promoters, CMV-LTR-hybrids, SV40 promotersor LTR-promoters e.g. from MMTV (mouse mammary tumor virus; Lee et al.(1981) Nature 214, 228-232), and additional viral promoter and activatorsequences derived from, for example, HBV, HCV, HSV, HPV, EBV, HTLV orHIV. One example for a controllable element that allows for acontrollable expression in eukaryotes is the tetracycline operator incombination with a corresponding repressor (Gossen M. et al. (1994)Curr. Opin. Biotechnol. 5, 516-20).

Examples for controllable elements that allow for the T-cell specificexpression in eukaryotes are promoters or activator sequences frompromoters or enhancers of those genes that code for proteins that areonly expressed in these types of cells.

Examples for controllable elements that allow for the cell cyclespecific expression in eukaryotes are promoters of the following genes:cdc25, Cyclin A, Cyclin E, cdc2, E2F, B-myb or DHFR (Zwicker J. andMüller R. (1997) Trends Genet. 13, 3-6). Examples for controllableelements that allow for the metabolically specific expression ineukaryotes are promoters that are regulated by hypoxia, by glucosestarvation, by concentration of phosphate or by heat shock.

The vector according to the invention can be used for the transfectionof a host cell that is preferably a T-cell. Particularly preferred is ahost cell which is characterized in that it expresses a polypeptide orfusion protein according to the invention on its surface. An additionalobject of the invention therefore relates to a method for producing apolypeptide for the diagnosis and/or treatment of diseases that arerelated to oncoproteins or for identifying pharmacologically activesubstances in a suitable host cell, which is characterized in that anucleic acid according to the invention is suitably expressed.

Thus, for example the polypeptide is produced according to methods thatare generally known to the person of skill, by expressing of the nucleicacid according to the invention in a suitable expressions system, asalready described above. As host cells, for example, the E. coli strainsDHS, HB101 or BL21, the yeast strain Saccharomyces cerevisiae, insectcell lines, e.g. from Spodoptera frugiperda or the animal cells COS,Vero, 293, HaCaT, and HeLa can be used, which are all commonlyavailable.

In order to allow for the introduction of nucleic acids according to theinvention and thereby the expression of the polypeptide in a eu- orprokaryotic cell by transfection, transduction, transformation orinfection the nucleic acid can be present as plasmid, as a part of aviral or non-viral vector or particle. Here, particularly suitable asviral vectors or particle are: baculoviruses, vacciniaviruses,retroviruses, adenoviruses, adeno-associated viruses, and herpesviruses. As non-viral carrier, in particular: virosomes, liposomes,cationic lipids or poly-lysine conjugated DNA are suitable.

Examples of gene-therapeutically effective vectors are viral vectors,for example adenoviral vectors or retroviral vectors (Lindemann et al.,1997, Mol. Med. 3: 466-76; Springer et al., 1998, Mol. Cell. 2: 549-58;Weijtens et al. “A retroviral vector system, ‘STITCH’; in combinationwith an optimized single chain antibody chimeric receptor gene structureallows efficient gene transduction and expression in humanT-lymphocytes”, Gene Therapy (1998) 5,1995-1203).

A preferable mechanism in order to bring about expression ofpolypeptides according to the invention in vivo is the viral genetictransfer, in particular with the aid of retroviral particles. These arepreferably used in order to provide respective target cells of thepatient, preferably T-lymphocytes, ex vivo with the genes or nucleotidesequences that encode for polypeptides according to the invention bytransduction. Subsequently, the target cells can be reinfunded into thepatient in the sense of an adoptive cellular transfer in order to takeover tumoricidal and/or immunomodulating effector functions with the denovo introduced specificity. Recently, using this way, very promisinggene-therapeutical successes in the treatment of SCID-X1-disease innewborn being characterized by an immune incompetence were achieved,wherein hematological precursor cells were retrovirally provided with ananalogous intact transgen of a non-functionally mutated variant of agene for the γ-chain present in infants that is essential for thedifferentiation in the different effector cells of the adaptive immunesystem (Cavazzana-Calvo et al., 2000).

Furthermore, the possibility exists to perform the genetic transfer invivo, one the one hand by preferentially stereotactic injection of theinfectious particles on the other hand by direct application ofvirus-producing cells (Oldfield, et al. Hum. Gen. Ther., 1993, 4:39-69).

The viral vectors that are commonly used for the transfer of genes inaccordance with the current state of the art are primarily retroviral,lentiviral, adenoviral and adeno-associated viral vectors. These arecircular nucleotide sequences that are derived from natural viruses,wherein at least the viral structural protein encoding gene has beenreplaced by the construct to be transferred.

Retroviral vector systems provide the condition for a long lastingexpression of the transgene by the stable but uncontrolled integrationinto the genome of the host. Vectors of the younger generation have noirrelevant and potentially immunogenic proteins, furthermore, there isno previous immunity of the acceptor against the vector. Retrovirusescontain an RNA-genome that is packaged into a lipid envelope thatconsists of parts of the cellular membrane of the host and viralproteins. For the expression of viral genes the RNA-genome is reverselytranscribed and integrated into the DNA of target cell with the enzymeintegrase. Then, the genes can be transcribed and translated by theinfected cell, whereby viral parts are generated that assemble to formretroviruses. Only at this time the RNA is introduced into the newlygenerated viruses. The genome of retroviruses has three essential genes:gag, that encodes for viral structural proteins, so-called groupspecific antigens, pol for enzymes such as reverse transcriptase andintegrase, and env for the envelope protein (“envelope”), that isresponsible for the binding of the host-specific receptor. Aftertransfection, the production of the replication incompetent virusestakes place in so-called packaging cell lines, that have beenadditionally provided with the gag/pol-encoding genes, and express these“in trans”, and therefore complement the formation of replicationincompetent (i.e. gag/pol-deleted) transgenic viral particles. Analternative is the co-transfection of the essential viral genes, wherebyonly the vector containing the transgenes carrying the packaging signal.

The separation of these genes on the one hand enables any combination ofthe gal/pol-reading frame with env-reading frames obtained fromdifferent strains, whereby pseudotypes with modified host tropism aregenerated, on the other hand thereby the formation of replicationcompetent viruses inside packaging cells can be drastically reduced. Theenvelope protein derived from “gibbon ape leukemia virus” (GALV) that isused in the “stitch” or “bullet”-vector system, respectively, has theability to transduce human cells, and is established in the packagingcell line PG13 with an amphotrophic host region (Miller et al., 1991).In addition, the safety is increased by a selective deletion ofnon-essential viral sequences for preventing a homologous recombinationand thus increases the production of replication competent-particles.

Novel non-viral vectors consist of autonomous non self-integratingDNA-sequences, the transposons, that are introduced by e.g. liposomaltransfection into the host cell, and have for the first time beensuccessfully employed for the expression of human transgenes inmammalian cells (Yant et al., 2000).

Gene-therapeutically effective vectors can be obtained by complexing thenucleic acid according to the invention with liposomes, since thereby avery high transfection efficiency, in particular of skin cells, can beachieved (Alexander and Akhurst, 1995, Hum. Mol. Genet. 4: 2279-85).Excipients, that increase the transfer of nucleic acids into the cell,can be, for example, proteins or peptides that are bound to DNA, orsynthetic peptide-DNA-molecules, that allow for the transport of thenucleic acid into the nucleus of the cell (Schwartz et al. (1999) GeneTherapy 6, 282; Brandén et al. (1999) Nature Biotech. 17, 784).Excipients also include molecules that allow for a release of nucleicacids into the cytoplasm of the cell (Planck et al. (1994) J. Biol.Chem. 269, 12918; Kichler et al. (1997) Bioconj. Chem. 8, 213) or, forexample, liposomes (Uhlmann and Peymann (1990) supra). Anotherparticularly suitable form of the gene-therapeutical vectors can beobtained by attaching the nucleic acids according to the invention ontogold particles, and shooting these into tissue, preferably into theskin, or cells with the aid of the so-called “gene gun” (Wang et al.,1999, J. Invest. Dermatol., 112:775-81).

For the gene-therapeutical use of the nucleic acid according to theinvention it is also advantageous if the part of the nucleic acidencoding for the polypeptide contains one or several non codingsequences, including intron sequences, preferably between promoter andthe start codon of the polypeptide, and/or a polyA-sequence, inparticular the naturally occurring polyA-sequence or an SV40 viruspolyA-sequence, in particular at the 3′-end of the gene, since thereby astabilization of the mRNA can be achieved (Jackson, R. J. (1993) Cell74, 9-14 and Palmiter, R. D. et al. (1991) Proc. Natl. Acad. Sci. USA88, 478-482).

An additional aspect of the present invention relates to a host cell,that contains a DNA- or RNA-vector molecule according to the invention.This in particular con be a T-cell that is transformed with a vectoraccording to the invention or another genetic construct according to theinvention. Host cells can be prokaryotic as well as eukaryotic cells,examples for prokaryotic host cells are E. coli and for eukaryotic cellsare Saccharomyces cerevisiae or insect cells.

Therefore, an additional aspect relates to a recombinant T-cell thatexpressed at least one mutated TCR according to the present invention. Aparticularly preferred transformed host cell is a transgenic T-precursorcell or a stem cell that is characterized in that it comprises a geneticconstruct according to the invention or an expression cassette accordingto the invention. Methods for transformation or transduction of hostcells and/or stem cells are well known to the person of skill, and, forexample, include electroporation or microinjection. A particularlypreferred transformed host cell is a patient-unique T-cell, that isafter the extraction transfected with a genetic construct according tothe invention. According to the invention, host cells in particular canbe obtained by extracting one or several cells, preferably T-cells, inparticular CD8⁺-T-cells that are subsequently transfected or transducedex vivo with one or more genetic constructs according to the invention,in order to thereby obtain host cells according to the invention. Then,the ex vivo generated specific T-cells can subsequently reimplanted intothe patient. The method therefore is similar to the method described byDarcy et al. (“Redirected perforin-dependent lysis of colon carcinoma byex vivo genetically engineered CTL” J. Immunol., 2000, 164:3705-3712) byusing scFv anti-CEA receptor transducec CTL, perforin, and γ-IFN.

The modified (poly)peptide and their derivatives according to theinvention for example can be used for the active and/or passiveimmunization of patients with diseases, in particular timorous diseasesthat for example are related to MDM2. A particularly preferred aspect ofthe present invention therefore relates to the use, wherein a cancerousdisease is treated, in particular a cancerous disease, that is relatedto a modified expression of MDM2, in order to achieve the induction,production, and increase of oncogen-specific, e.g. MDM2-specific CTL,and to specifically kill the tumor- and leukemic cells of the respectivepatient. Such diseases, for example, include solid timorous diseases,lymphohematopoeitic neoplasia, malign hematological diseases, also inform of a multiple myeloma (or plastocytoma), a histiocytic lymphoma anda burst of CML-blasts. Respective related TAAs against which thecorresponding TCRs can be developed are, for example, p53, Her-2/neu,Ras, tyrosinase, MART, Gp100, MAGE, BAGE, MUC-1, CD45, CD19, andPRDI-BF1.

A particularly preferred aspect of the present invention therefore inaddition relates to: a composition, in particular a pharmaceuticalcomposition that comprises a recombinant T-cell according to the presentinvention. Furthermore, preferred is the use of a mutated alpha- orbeta-chain of a TCR according to the present invention, of a mutated TCRaccording to the present invention, and/or a recombinant T-cellaccording to the present invention for the production of therapeuticsand/or prophylactics for the treatment of cancerous diseases. In aparticularly preferred manner of the treatment, one or more cells,preferably T-cells, in particular CD8⁺-T-cells are extracted from thepatient that are subsequently transduced or transfected ex vivo with oneor several genetic constructs according to the invention. Then, the exvivo generated specific T-cells subsequently can reimplanted into thepatient. The composition according to the invention furthermore canfurther contain suitable additives and excipients.

An object of the present invention is also a medicament for theindication and therapy of diseases associated with oncoprotein-protein,containing a nucleic acid according to the invention or a polypeptideaccording to the invention and, optionally, containing suitableadditives and excipients, as well as a method for producing of such amedicament for the treatment of with diseases associated withoncoprotein-protein, wherein a nucleic acid according to the inventionor a polypeptide according to the invention is formulated with apharmaceutically acceptable carrier. As therapeutics and/orprophylactics in particular vaccines, recombinant particles or solutionsfor injection or infusion can be considered that contain (a) theTCR-receptor polypeptide according to the invention and/or itsderivatives and/or (b) a nucleic acid according to the invention asactive ingredient, and/or (c) T-lymphocytes generated in vitro or exvivo that contain a specific mutated TCR being directed againstOncoprotein.

For the gene therapeutical use in humans in particular a medicamentand/or recombinant particle is suited that contains the nucleic acidaccording to the invention in naked form or in form of one of the abovedescribed gene-therapeutically effective vectors or in a form complexedwith liposomes or gold particles respectively. The pharmaceuticalcarrier, for example, is a physiological buffer solution, preferablyhaving a pH of about 6.0-8.0, preferably of about 6.8-7.8, in particularof about 7.4 and/or of an osmolarity of about 200-400 milliosmole/litre,preferably of about 290-310 milliosmol/litre. In addition thepharmaceutical carrier can contain suitable stabilizators, such as, forexample, nuclease inhibitors, preferably complexing agents such as EDTAand/or other excipients known to the person of skill.

The term “coding nucleic acid” refers to a DNA-sequence encoding for aisolatable bioactive polypeptide according to the invention or aprecursor. The polypeptide can be encoded by a sequence in its completelength or any part of the coding sequence as long as the specific, forexample, enzymatic activity is preserved.

It is known that small modifications can be present in the sequence ofthe nucleic acids according to the invention, for example by thedegeneration of the genetic code, or that non-translated sequences canbe attaches to the 5′ and/or 3′-end of the nucleic acid, without thatits activity will be essentially modified. Therefore, this inventionalso encompasses so-called “functional variants” of the nucleic acidsaccording to the invention.

With the term “functional variants” all DNA-sequences are designatedthat are complementary to a DNA-sequence that hybridizes under stringentconditions with a referenz sequence derived therefrom or parts thereof,in particular the hypervariable V(D)JC-region, and have activity that issimilar or identical to the corresponding polypeptide according to theinvention.

By “stringent hybridization conditions” those conditions are to beunderstood, wherein a hybridization occurs at 60° C. in 2.5×SSC-buffer,followed by several washing steps at 37° C. in a lower bufferconcentration and maintains stable.

With the term “functional variants” in the sense of the presentinvention polypeptides are to be understood that are functionallyrelated to the polypeptides according to the invention, i.e. havestructural features of the polypeptides. Examples of functional variantsare the corresponding polypeptides that are stemming from otherorganisms as the mouse, and also human, preferably from non-humanmammals such as, for example, monkeys, porcine and rat. Other examplesof functional variants are polypeptides that are encoded by differentalleles of the gene in different individuals or in different organs ofan organism. Encompassed by the present invention are, in particular,also functional TCR-variants that recognize the identical epitope of theMDM2-polypeptide and trigger a specific T-cell response.

In a further sense also polypeptides are to be understood that have asequence homology, in particular a sequence identity, of about 70%,preferably about 80%, in particular about 90%, more particularly about95% to the polypeptides having the amino acid sequence according to oneof the SEQ ID No. 1 to SEQ ID No. 6 and/or to the DNA sequences that arederived from the peptide sequences. Among those are also additions,inversions, substitutions, deletions, insertions or chemical/physicalmodifications and/or exchanges or parts of the polypeptide in a size ofabout 1-60, preferably of about 1-30, in particular of about 1-15, moreparticularly of about 1-5 amino acids. For example, the first amino acidmethionine can be missing without that the function of the polypeptideis essentially modified.

The invention shall now be further explained based on the accompanyingExamples and Figures without being limited by these.

SEQ. ID No. 1: shows the alpha-chain of the murine TCR 1(1TCR_aTCR.pro),

SEQ. ID No. 2: shows the alpha-chain of the murine MDM2 TCR(Mu_Wt_aTCR_MDM2.pro),

SEQ. ID No. 3: shows the alpha-Chain of the human TCR 1bd2 (1bd2_a.pro),

SEQ. ID No. 4: shows the beta-Chain of the murine TCR 1 (1TCR_bTCR.pro),

SEQ. ID No. 5: shows the beta-Chain of the murine MDM2 TCR(Mu_Wt_bTCR_MDM2.pro), and

SEQ. ID No. 6: shows the beta-Chain of the human TCR 1bd2 (1bd2_b.pro).

FIG. 1 shows the superposition of the protein backbone of proteincrystal structures of a murine H2-K^(b)-restringated (1, Garcia et al,1998) and a human HLA-A2-restringated (1bd2, Ding et al., 1998)T-cell-receptor. The heterodimeric human TCR is depicted in dark grey(αTCR) and light gray (βTCR) for the respective chains, the murine TCRfor the single chains is gray in total.

FIG. 2 shows the representation of the protein backbone of 1, whitexclusively those side chains that are identical to 1bd2. The uppergraphics illustrates the presence of few identical residues in thecontact area of the variable domains (Vα, Vβ) of both chains. The lowergraphic is turned around the axes in the paper-plain in such a mannerthat it documents the numerous presences of identical amino acids in thecontact area of the constant domains (Cα, Cβ).

FIG. 3 shows the representation of relevant MDM2(81-88)-specificTCR-constructs, from which in the murine model that is presented therespective wild type chains (Wt) of the Mu Wt TCR MDM2 were mutated(Mut) in correspondence with the graphics, and were combined in murineT-cells after retroviral transfer. The murine variable domains arewhite, the constant domains are hatched in gray. The partially humanizedTCR (Mu Chim TCR MDM2) exhibits a tighter gray hatching in the constantdomain. Below, a single chain TCR is depicted whose variable domains arelinked via a (GlyGlyGlyGlySer)₃-linker. The length of the cylinderindicated the relevant amino acid positions and their sterical sizes.The yellow arrow symbolizes the absence of the interaction of the chainsdue to sterical interference (Mu Mutα/Wtβ TCR MDM2) or the absence ofinteraction (Mu Wtα/Mutβ TCR MDM2) of the respective pairs of aminoacids.

FIG. 4 shows the amino acid sequence alignment of the murine TCR1(1TCR_aTCR.pro or 1TCR_bTCR.pro respectively), for which the structuraldata was present with the MDM2-specific TCR (Mu_Wt_aTCR_MDM2.pro andMu_Wt_bTCR_MDM2.pro, respectively) and the human TCR 1bd2, for whichlikewise structural date was present. The heights of the bars indicatethe extent of the identity as found at the respective positions. Fromthese, it can be taken that the amino acids that are immediatelysurrounding the mutated amino acids and largely are interactingtherewith are mostly conserved between human and mouse. The analysis ofthe crystal structures indicated insignificant differences. FIG. 4 a:α-chains-comparison; FIG. 4 b: β-chains-comparison.

FIG. 5 shows the indication of the amino acid pairs in the structurallywhole context of 1, corresponding to the mutated amino acid pairGly¹⁹²/Arg²⁰⁸ of MDM2-specific TCR. The amino acids are intermediatelypositioned in the drilled β-sheets of both chains within the constantdomains that are wound one around the other and are aligned one to eachother. For orientation, the relevant CDR3-loops of the variable domainsthat recognize the peptide antigen of 1 are shaded in a diverging shadeof gray: the affected pair of amino acids is positioned far away fromthe region that is responsible for the binding of theMHC-peptide-complexes.

FIG. 6 shows the sterical depiction of the wild type amino acid pairGly¹⁷⁹/Arg¹⁹⁵ (FIG. 6 a) and the mutated amino acid pair Arg¹⁷⁹/Gly¹⁹⁵(FIG. 6 b) of the 1.pdb as can be found for the latter followingstructural data-supported design. FIG. 6 a includes those amino acids ofthe central β-sheets having large sterical side chains, in contrast FIG.6 b includes those amino acids in a spherical region of a diameter of 5Angstroms around the Cα of the Arg¹⁷⁹ that was mutated on the screen byomitting those side chains that point away from the contact region ofthe chains (for reasons of simplicity). A conformer was chosen that doesnot have an affecting interaction with the neighboring side chains. Thestretched conformation enables the formation of an H-bridge to thecorresponding main-chain-oxygen of the β-chain in near analogy to thesituations in wild type (FIG. 6 a).

FIG. 7 shows the FACS-analysis of the human transduced T-cells that eachwas provided with the different combinations of the TCR-constructsdescribed in FIG. 3. Depicted is a 2-fold staining of vβ6-FITC andCD8-PC5: only CD8-positive transduced T-cells show the desired cytotoxiceffector function. The vβ6-staining allows for the determination ofβ-chain but not for the α-chain.

FIG. 8 shows the FACS-analysis of human transduced T-cells that each wasprovided with the different combinations of the TCR-constructs asdescribed in FIG. 3. Depicted is a 2-fold staining of TetMDM2-PE andCD4-FITC: the tetramer-staining enables the determination of functional,heterodimeric αβTCR, thus the indirect determination of the α-chain.Only CD8-positive T-cells can be stained since the tetramer in case ofmoderately to highly affine binding TCR is binding dependent from CD8.

FIG. 9 shows the cytotoxicity-assay (Stanislawski & Voss et al, 2001)with the combined double-chain TCR shown in FIG. 3: TAP-deficient T2were loaded exogenously with the indicatedMDM2(81-88)-peptide-concentrations and tested for recognition by thetransduced T-cells: the extend of lysis is reflected in the quantity bythe ⁵¹Cr that was taken up into the cells and released by lysis. Agp100-dereived peptide functioned as a relevant peptide. The “effector:target”-Ratio as well as the corrected CD8⁺vβ6⁺-ratio are given sincethese varied between the different transduction-approaches.

FIG. 10 shows the cytotoxicity-assay (Stanislawski & Voss et al, 2001)with the combined double-chains TCR shown in FIG. 3: HLA-A2-positiveleukemia-cell lines of different origin (ATCC (American Type CultureCollection, Manassas, USA), DSMZ (Deutsche Sammlung von Mikroorganismenand Zellkulturen GmbH, Braunschweig)), that over expressed MDM2 andprocess the MDM2(81-88)-peptide were used as “target”. The A2-negativeleukemia-cell line UocB1 and the MDM2-negative cell line Saos2 served asnegative control. A MDM2-transfectant of Saos2, Saos2 c16, was alsospecifically recognized.

FIG. 11 shows the different possibilities of the inversion of stericrelations, the charges or both, according to the method according to theinvention.

EXAMPLES

point mutants (Mut) of the murine wild type-TCR (Wt) were to bedetermined that fulfill the above described “knob-hole”-model: a pairingof chains should occur only those TCR-chains that carry each one of thesterically inverted amino acid partners, whereas the combination of wildtype and mutated chain in both conceivable orientations should beaffected (FIG. 3). In addition, the combined TCR had to be tested afterintroduction into human T-cells in view of their structural avidity,i.e. their structural integrity and in view of their functional avidity,i.e. the maintenance of the peptide-dependent effector function (Bullocket al., 2001). The MDM2(81-88)-specific T-cell-receptors as describedabove and identified in our laboratory whose amino acid sequence of theconstant domains is nearly identical to the murine TCR 1.pdb for whichstructural data is present (FIG. 4 a/b, Garcia et al., 1998) and thatexhibits a high homology in the variable domains to this, was used as amodel system. MDM2 is a human regulatory proto-oncoprotein that counterregulates the expression of the tumor suppressor protein p53(Stanislawski & Voss et al., 2001).

The fashion of the selection of the point mutations occurred after theanalysis of published TCR-crystal structures and the comparison ofhomology of murine and human sequences. The point mutations were thenintroduced into the murine TCRs being similar to the human, andexperimentally functionally tested. It can be derived from the homologycomparison against human sequences that the structural situations inhuman TCRs in the intermediate surroundings of the amino acid exchangesare nearly identical and therefore a pairing of “mutated murine chainwith humane wild type-chain” in the configuration “αTCR-βTCR” or“βTCR-αTCR” must be likewise reduced.

The point mutants so far are not yet absolutely functional, neverthelessinterfere noticeably with the formation of unwanted pairs. These aminoacid exchanges follow the general “knob-hole”-model which indicates toinvert the proportions of interacting amino acids in order to introducea chain-specific interaction. Further mutations can be provided in thechains as already generated in order to further increase thespecificity. This aspect is also included in the scope of the presentinvention.

The selection of point mutants was made in such a manner that these, atleast for different murine TCR of different peptide-specificity could begeneralized. In addition, these point mutants can also be applied tohuman TCRs since the murine TCR in a parallel project, by maintainingthe peptide-effector-function, should be maximally humanized in order toavoid immune reactions against the exogenic TCR. Therefore,preferentially identical amino acids versus homologous amino acidsshould be selected. A sequence comparison of several murine and humanTCR-sequences showed an exceedingly high homology in the constantdomain, whereas the variable domain exhibited only low homologies (FIG.4 a/b). There are numerous amino acids that are conserved in the contactarea of the heterodimeric chains between human and mouse, and had to beindividually verified for the solution of the problem (FIG. 2). As anessential criterion, the amino acids as selected, must be integrateableinto the “knob hole”-model, i.e. an amino acid having a correspondinglylarge side chain must interfere with a smaller amino acid of the otherchain. Therefore, it was a priori irrelevant in which of both chains thelarge amino acid was present. As large amino acids, tryptophane, lysine,arginine, phenylalanine and tyrosine could be taken into account, assmall, in particular, glycine, serine, and alanine. The directlyadjacent amino acids should, in case of the potential inversion of thesteric situations at the respective position, should behave as inert aspossible, i.e. should not have a pronounced interaction whetherhydrophobic or charged nature with the interacting pair of amino acids.

For the examination of murine TCR-structures the coordinative data ofthe murine TCR 1.pdb (Garcia et al., 1998) was downloaded from the“Brookhaven Protein Database” (www.resb.org/pdb) charged and visualizedby means of the structure-depicting software “Swiss-PDBViewer”(www.expasy.ch/spclbv).

The following position was found to be particularly attractive: thearginin¹⁹⁵ of the βTCR from 1.pdb, in a nearly stretchedall-trans-conformation of a side chain, pointed in the direction of theαTCR (FIG. 5). Compared to the guanidinium-Group of Arg¹⁹⁵ Gly¹⁷⁹ of theαTCR is present in an ideal Van der Waals-distance. The amino acidsequence on the side of the α-chain around Gly¹⁷⁹ is

The opposing β-strand of the β-chain that contains the Arg¹⁹⁵ isanti-parallel and approximately twisted by 30° to the β-strand of theα-chains: through this the side chains of the neighboring β-strands comeinto the sterical vicinity to the interacting pair of amino acids. Theβ-chains-amino acid sequence at this position is identical between humanand mouse, the one of α-chain is largely homologous: Gly¹⁷⁹ is replacedby the likewise small amino acid serine in human, Ile¹⁸¹ by thehomologous valine. The sequences of the β-strands of both chains thatare neighboring on the same level to the Gly¹⁷⁹/Arg¹⁹⁵ are identicalover a length of 5 amino acids between human and mouse and are thereforealso the potential interacting partner immediately around Gly¹⁷⁹/Arg¹⁹⁵(FIG. 4 a/b).

At best, weak hydrophobic interactions of neighboring side chains mustbe considered fort he pair of amino acids. The contact areas of chainsat this position is not densely packed: only few long chain amino acidspoint into the direction of the opposing chain or offer extensivehydrophobic Van der Waals-contacts. This again stresses the structuralmeaning of the projecting side chain of the Arg¹⁹⁵. The pair of aminoacids is intermediately localized in a drilled β-sheet of the constantdomain being partially wound around itself of both chains (FIG. 6 a/b):each β-sheet consists of four β-strands out of which an intermediateβ-strand contains one of the targeted amino acids, respectively. Eachβ-sheet of the individual chain for itself forms numerous H-bridges butnone of them via their amino acid side chains towards the opposingchain. Exclusively Arg¹⁹⁵ by the terminal nitrogen of theguanidinium-group forms two H-bridges towards the opposing main chainoxygen and side chain oxygen of Thr¹⁶⁶ of the α-chain. A central bridgeis formed in a contact region that is apart from that characterized byfew salt bridges and dipole-dipole interchanging effects as well as by alow packing of hydrophobic amino acids. At the edge of the cavity to beformed (FIG. 6 a) there are numerous contacts between the chains,nevertheless those are not comparably well positioned in the middle ofthe contact area of the respective wound β-sheets. The stretchedconformation of the Arg¹⁹⁵ as well as the low hydrophobic packing enablean inversion of the amino acids without essentially affecting the localstructure. A mutated Arg¹⁷⁹ of the αTCR would likewise be able to atleast form an H-bridge towards the main chain oxygen of Ser¹⁷³ of theopposing β-strand of the βTCR without that the mutated Gly¹⁹⁵ wouldprovide any interference (FIG. 6 b). A slight positive charge shift ofthe guanidinium-group from the α-chain in the direction of the β-chainresults. The amino acids do not have to be qualitatively modified inorder to generate comparable steric (i.e. ideal hydrophobic distances)and polar relations (i.e. H-bridges). The positions inMDM2(81-88)-specific TCR that are identical to Arg¹⁹⁵ and Gly¹⁷⁹ of1.pdb are Arg²⁰⁸ and Gly¹⁹². In human T-cell-receptors the arginine ofthe β-chain is conserved, whereas the likewise small amino acid serineis present at the position of the glycine of the α-chain (1bd2; FIG. 4).A publication about mutated or truncated TCR proves the importance ofthe constant domain for the pairing of chains by Coulomb-forces betweencharged amino acids residues of the region Ser¹⁸⁸-Leu²¹³ of the β-chainwherein the point mutant is positioned that is described by theinventors (Li et al., 1996).

The corresponding mutation are introduced in the respectiveMDM2(81-88)-specific TCR-genes that were already individually present inthe retroviral vector pBullet (Willemsen et al., 2000) with the aid ofthe “Quikchange™ Site-Directed Mutagenesis”-Kit (Stratagene)(Stanislawski & Voss et al., 2001).

The adoptive transfer into human T-cells occurred principally asdescribed in Stanislawski & Voss et al (2001). The co-transfectionsystem (Weijtens et al., 1998) that provides a co-transfection ofindividual plasmids with each of a chain of the heterodimeric TCRencoded as transgene, enables the combination of all conceivable wildtype and mutated TCR-chains. The wild type TCR versus the TCR beingmutated in both chains versus a TCR being mutated in only one chainshould be analyzed structurally by FACS-analysis and functionally bycytotoxic lysis of antigen-presenting cells (APC) as lyseable targetcells. Therefore, in the following designated “hybrid TCR” being mutatedin only one chain serves as a model for the “unwanted” TCR-pair ofchains from mutated exogenic murine α- or β-chain and the wild typeendogenic human β- or α-chain (FIG. 3), as could be hypotheticallypresent in case of an adoptive transfer in human T-cells. This could beassumed since the structural backbone as well as the amino acidsequences of humane and murine TCR in the constant domain are stronglyconserved and can potentially interact and mutations will exhibit acomparable effect on pairing of chains and antigen recognition. Hereby,the endogenic TCR being present in the human T-cells did not interferewith the analysis of different murine TCR-combinations since themutations of the murine chains could at best have a parallel effect onthe pairing of chains to the human “pendant”. In addition, it isimportant to perform these experiments in an experimental design that isas similar as possible to the clinical application.

In order to start with the assumption that all T-cells contain thetransgene, after transduction the T-cells were selected by G418(selection marker for the β-chain that is following the transgene via anIRES-element) as well as via puromycin (selection marker for theα-chain). Thus, only those T-cells survive that produce the bicistronicmRNA from βTCR-transgene and G418-marker as well as the bicisronic mRNAfrom αTCR and puromycin. Differences in the FACS-staining therefore donot result from differences in transduction efficiency from the humanT-cells but are reflecting the respective TCR-stability.

The structural avidity as an expression of stable expression of the wildtype as well as the mutated TCR was analyzed on the one hand via thesub-family of the specific staining of β-chain (vbeta6-FITC; FIG. 7) aswell as the TCR-specificity distinguishing staining by means ofMDM2(81-88)-specific TCR-tetramers (Klenerman et al., 2002, FIG. 8) bymeans of FACS-analysis. The tetramers were produced in the laboratory ofDr. Pedro Romero (University Lausanne, Switzerland), and provided forscientific purposes.

It could already be seen from the vβ6-staining that the “hybrid” TCRs,Mu Mutα/Wtβ TCR MDM2 and Mu Wtβ/Mutα TCR MDM2, did express the α-chainat least as unstable as was known from a partially humanized TCR, MuChim TCR MDM2 (FIG. 7). A further indicator of a prominentTCR-instability was the tetramer-colorability, which was nearly missing(FIG. 8). The TCRs that were mutated in both chains, Mu Mutα/Mutβ TCRMDM2, were determined in the vβ6- as well as the tetramer-staining in anearly comparable fashion to the murine wild type-chains, Mu Wt TCRMDM2.

The efficiency of lysis as a measure of the functional avidity wasmeasured in the chrome-release assay or cytotoxicity assay. For this,the cell line T2, that is unable to load endogenically processedpeptides onto MHC-molecules and to transport the complexes to thecellular surface, was exogenically loaded with the MDM2(81-88)-peptidein a concentration dependent manner, and the half maximal lysis as ameasure of the recognition was measured in a peptide titration (FIG. 9).The different vβ6- as well as CD8-positivity of the differentlytransduced T-cell-populations were indicated by stating theCD8⁺vβ6+:T-ratios in addition to the common E:T(effector:target)-ratios: differences in the efficiency of lysistherefore reflect the differently combined TCR-constructs throughcorrecting both T-cell-phenotype-markers as an expression of the percentstrength of expression of the βTCR (vβ6+) and the percent strength ofthe cytotoxic T-cell-population (CD8⁺). Congruent with the data of thestructural avidity it could be taken from the functional data that the“hybrid” TCRs were markedly affected in the efficiency of lysis comparedto the wild type, and still were worse than the chimeric partiallyhumanized TCR, although in these a complete domain (Cα or Cβ,respectively) and not only one amino acid was exchanged like in the“hybrid” TCRs. The TCR being mutated in both chains mutated TCR,Mutα/Mutβ TCR MDM2, exhibited only slightly worsened efficiencies oflysis compared to the wild type.

These quantitative differences then should be examined whether there isa critical half maximal lyses, i.e. a threshold at which target cellsthat endogenically present the respective peptide, are first recognized.For this, different target cells were examined in a cytotoxicity assay(FIG. 10): here, Saos2 serves as a negative control of theMDM2-expression, whereas Saos2 c16 represents a MDM2-transfectant withpositive MDM2(81-88)-processing. The leukemia-cell line UocB1 isHLA-A2-negative and indicates the MHC-restriction of the transducedT-cells. The other leukemia-cell lines EU-3, BV-173 and IM-9 are ofdifferent origin (ATCC, DSMZ) and prove the generalization of theMDM2(81-88)-recognition by the transduced human T-cells. Also in thiscase it could be shown that the TCR being mutated in both chains iscomparable to the wild type and better than the chimeric TCR inrecognizing the endogenic “targets”. Although the “hybrid” TCR halfmaximally lyses the exogenically loaded cell line T2 up to 1 nM peptide(FIG. 9), these to not recognize the malign cell lines: obviously, thepeptide presentation of MDM2(81-88) has dropped below a critical value,below which this is no longer recognized. Saos2 c16 is weaklyrecognized, which most likely can be explained with the heterologous,promoter-driven strong expression of MDM2.

The data of the structural and functional avidity therefore arecongruent and prove the effectiveness of the selected point mutants in amurine TCR-model in human T-cells in order to drastically impairunwanted “pairing” of heterodimeric “hybrid” αβTCR.

Transduction of Human Peripheric Blood Lymphocytes (PBLs)

For the transduction of human peripheric blood T-lymphocytes, afunctional derivative of the pStitch-system (Weijtens et al., 1999) wasused. The retroviral genes required for packaging were encoded byindividual plasmids by way of a co-transfection of the packaging cellline 293T (Soneoka et al., 1995): pHit60 encodes for thegag-pol-structure- and polymerase-gene from the Moloney murine leukemiavirus (MoMuLV), pColt-Galv for the env-envelope protein of the “gibbonape leukemia virus”, that is able to bind to theNa⁺-/phosphate-synporter pit of human cells, and thereby to transducethe latter. The chimeric viral particles therefore exhibit anamphotrophic pseudotype, and are able to transduce different mammaliancells, apart from mouse.

Transfection of the Packaging Cell Line 293T

The isolated bacterial clone of the T-cell-receptor-genes cloned in thepStitch-derivative were purified by means of plasmid-preparations, thatassures a removal of residual endotoxines (Qiagen, product 12362), andadjusted to 1 μg/μl. The DNA was transiently introduced into thepackaging cell line 293T (GiboBRL-Life Technologies, product 18306-019)via the calcium phosphate-precipitation. Here, in the context of themodified T-cell-receptors, αTCR and βTCR up to 80 μg DNA are employed:

-   -   20 μg αTCR-construct    -   20 μg βTCR-construct    -   20 μg pColt-Galv    -   20 μg pHit 60

In case of single chain-TCRs 60 μg DNA are used. 293T were grown in amodified DMEM-medium (DMEM/H):

-   -   DMEM, 4.5% glucose (BioWhittaker)    -   10% heat inactivated FKS    -   2 mM glutamine    -   1× penicillin/streptomycin    -   1× non-essential amino acids    -   25 mM HEPES

On the day before the transfection, the 293T cells were seeded at0.9*10⁶ cells per T25-flask and transfection approach in 5 ml DMEM/H. 4h before transfection the medium was replaced with fresh DMEM-H (3 ml)that was warmed to room temperature (RT). The transfection occurredaccording to the instruction of the commercial protocol (Invitrogen). 1ml of the transfection approach was pipetted into the respective flaskby careful drop wise addition. The DNA-Ca₃(PO₄)₂-precipitate shouldspread out finely distributed onto the adherent cells.

At the following morning, the medium was replaced with fresh DMEM/Hwhich was warmed to room temperature. 6 h later, the co-cultivation withthe activated PBLs took place.

Transduction of the Activated PBLs-Activation of Peripheric BloodLymphocytes (PBLs)

3 days before the scheduled co-cultivation ficoll-treated PBLs wereseeded at 2*10⁶ cells/ml in huRPMI-P in each 2 ml of a 24 well-plate(cellular tissue-treated surfaces). The activation occurred via thecross-linking antibody OKT-3 (Orthoclone-Diagnostics) at 20 ng/ml.

-   -   huRPMI-P:    -   RPMI 1640 (2 mM glutamine) without phosphate (Life Tec.,        11877-032)    -   10% human, heat inactivated AB-serum (HLA-A2.1 seropositive)    -   25 mMHEPES    -   1× penicillin/streptomycin (Life Tec.)

The plates were incubated in an incubator at 37° C. and 5% CO₂.

Co-Cultivation

For co-cultivation, the activated PBLs from the respective wells of a24-well-plate were pooled and counted. Adherent monocytes werediscarded. The cells were centrifuged (1500 rpm, 5 min, RT) andresuspended in a concentration of 2.5*10⁶ cells/ml in fresh huRPMI-P,and put back into the incubator. Prior to this, the medium was adjustedto 400 U/ml IL-2 (Chiron) and 5 μg/ml polybren (Sigma).

Each transfection approach was consecutively trypsinated 6 h after thechange of medium: for this, each T25 was washed with 3 ml HBSS (LifeTechnologies), incubated with 1 ml trypsin-EDTA (Life Technologies) formaximal 5 minutes, the dissolved cells were quantitatively taken up, andadded dropwise while stirring to 4 ml prepared huRPMI-P (RT). The 293Tcells were irradiated with 2500 rad. They were centrifuged (1500 rpm, 5min, RT) and resuspended in 4 ml fresh, adjusted huRPMI-P, supplementedwith 400 U/ml IL-2 and 5 μg/ml polybren. 1 ml of the adjusted PBLs wereadded to the preparation, and the preparation (0.5*10⁶ PBLs/ml) wasincubated for three days in an incubator (37° C., 5% CO₂).

On day 3 following co-cultivation, the suspended PBLs were taken up andresuspended in fresh medium huRPMI-P, supplemented with 40 U/ml IL-2(Chiron) and 2.5 μl CD3/CD28-beads, at 1*10⁶ cells/ml. 3 days later, ananew split in fresh medium took place. Within these 7 days, it wasmaximally expanded to the transition on T75-flasks. These cells could bedirectly employed in an immunological staining (FACS-analysis) or in aclassical ⁵¹chromium release assay.

Examples of the FACS-Analysis

Following the retroviral transduction, the above described constructswere analyzed in “fluorescence activated cell sorting” (FACS). For this,0.25*10⁶ cells were saturating stained with fluorophor-labelledantibodies: the heterologous expressed mutated β-chain was detected withanti-vp6-FITC (BD); the total of the T-cells by the marker anti-CD3-PC5(Coulter-Beckman). A sample that was transduced with the emptypStitch-derivative served as a negative-control. The expression could bereproduced in several donors of HLA-A2-positive T-cells. For thetetramer-staining, 5 μl of a 0.28 mg/ml stock solution for 45 min at 8°C. were used.

Cytolytic Activity of the Transduced T-Cells

The transduced T-cells were analyzed for their cytotoxic specificity ina classical ⁵¹chromium-release assay. In this system target cells wereradioactively labeled by the incorporation of ⁵¹chromium. If theretrovirally modified effector cells peptide-specifically recognized thetarget cell, the latter was driven into apoptosis by the effectorfunctions of the T-cell, and killed by lysis. The extent of the releasedchromium-nuclide allows for a conclusion about the effectiveness of thecellular recognition and lysis. The affectivity was tested over a broadrange of the ratio of tested effector cells to target cells (E:T) thatwere used. A murine MDM2-81-88 peptide specific T-cell-clone served as areference, from which the T-cell receptor gene was isolated. Targetcells were:

T2: human TAP-deficient cell line, which was to be loaded exogenouslywith any peptide. The specifically mutated peptide was MDM2-81-88, anirrelevant control-peptide was derived from the influenza-matrix proteinFluM 1.

Saos-2/6: transfectant of the human osteosarcoma Saos-2-cell line thatheterologously expresses and endogenously processes MDM2.

UocB11, EU-3: pre-B-cell-leukemia

IM-9: plasmocytoma

BV173: pre-B-cell-leukemia

1. A method for producing a heterodimeric specific wild type- orchimeric T-cell receptor (TCR), containing a first chain and a secondchain that interact one with another at at least one surface, whereinthe at least one surface is subjected to a rational mutagenesis, suchthat the at least one surface of the first chain or the surface of thesecond chain comprises a sterically projecting group, which interactswith a sterically recessed group on the at least one surface of thecorresponding first chain or second chain, comprising the steps of: (a)providing DNA-molecules, comprising the coding regions for the at leastone surface to be mutated of the first chain or second chain, in (a)joint or separate mutagenesis-vector system(s), (b) mutagenesis of theDNA-molecules, wherein the nucleic acid sequence encoding for the atleast one surface is modified compared to the initial sequence in such amanner that in the at least one surface of the first chain or the atleast one surface of the second chain, a sterically projecting group isintroduced, and in the corresponding at least one interacting surface ofthe second chain or the first chain, a sterically recessed group isintroduced, whereby individual mutated fragments are produced, and c)translation of at least two of the single mutated fragments from stepb), such that the pairing of the heterodimeric specificfirst-chain/second-chain TCR being mutated at least one surface isselectively promoted, and d) presentation of the heterodimericfirst-chain/second-chain TCR by a T-cell.
 2. The method according toclaim 1, wherein step c) is replaced by the following steps: (c′)optionally, sub-cloning of the mutated fragments into a suitabletransfection-vector system, (c″) transfection or co-transfection ortransduction of at least two of the mutated fragments into a mutantTCR-deficient T-cell, and (c′″) expression of the heterodimericfirst-chain/second-chain TCR in a recombinant T-cell.
 3. The methodaccording to claim 1, wherein step c) is replaced by the followingsteps: c′) In vitro-translation or in vivo-translation of at least twoof the individual mutant-fragments from step b) and, optionally,subsequent isolation and/or purification of the translatedmutant-fragments, such that the pairing of the heterodimeric specificfirst-chain/second-chain TCR being mutated at least on one surface isselectively promoted, and c″) introduction of the mutated specificfirst-chain/second-chain TCR into a T-cell.
 4. The method according toclaim 3, wherein the in vivo translation takes place in a host cell. 5.The method according to claim 3, wherein the introduction takes place byliposome-transfer.
 6. The method according to claim 1, wherein the TCRis an alpha/beta TCR, gamma/delta TCR, a humanized or partiallyhumanized TCR, a TCR being provided with additional (functional)domains, or a TCR being provided with alternative domains.
 7. The methodaccording to claim 1, wherein the amino acids as introduced after themutagenesis of the DNA-molecules are further suitably chemicallymodified, in order to thereby introduce a sterically projecting group ora sterically recessing group.
 8. The method according to claim 1,wherein the amino acids as introduced after the mutagenesis of theDNA-molecules directly provide the sterically projecting group or thesterically recessing group.
 9. The method according to claim 1, whereinthe amino acids as introduced by the mutagenesis of the DNA-moleculesare chosen in such a manner that a mutual exchange of the amino acids onthe surfaces of the interacting chains of the TCR is achieved.
 10. Themethod according to claim 1, wherein an amino acid that has beenintroduced after the mutagenesis of the DNA-molecules that introduces asterically recessing group compared to the initial sequence is selectedfrom glycine, serine, threonine, valine and alanine.
 11. The methodaccording to claim 1, wherein an amino acid that has been introducedafter the mutagenesis of the DNA-molecules that introduces a stericallyprojecting group compared to the initial sequence is selected fromtryptophane, lysine, arginine, phenylalanine, cysteine and tyrosine. 12.The method according to claim 1, wherein at least two surfaces of aTCR-chain are simultaneously subjected to mutagenesis.
 13. The methodaccording to claim 1, wherein the corresponding interacting surfaces arelocalized in the variable domains of the TCR-chains.
 14. The methodaccording to claim 1, wherein the corresponding interacting surfaces arelocalized in the constant domains of the TCR-chains.
 15. The methodaccording to claim 1, wherein the domains of the TCR-chains to bemutated are selected from mammalian domains.
 16. The method according toclaim 1, wherein the rational mutagenesis of the TCR-chains at the sametime leads to a humanization of the TCR.
 17. The method according toclaim 1, wherein the alpha- and beta-chains of an MDM2(81-88)-specificTCR are used as alpha-chain and beta-chain, and wherein the Gly192 ofthe constant region of the alpha-chain and the Arg208 of the constantregion of the beta-chain are exchanged by Arg 192 in the constant regionof the alpha-chain and by Gly208 in the constant region of thebeta-chain.
 18. The method according to claim 17, wherein simultaneouslywith or subsequent to the exchanges at positions 192 and 208, additionalpositions are modified in the chains.
 19. The method according to claim1, wherein a retroviral vector is used as a transfection system.
 20. Amutated alpha- or beta-chain of a TCR, produced according to a methodfor producing a heterodimeric specific wild type- or chimeric T-cellreceptor (TCR), containing a first chain and a second chain thatinteract one with another at at least one surface, wherein the at leastone surface is subjected to a rational mutagenesis, such that the atleast one surface of the first chain or the surface of the second chaincomprises a sterically projecting group, which interacts with asterically recessed group on the at least one surface of thecorresponding first chain or second chain, comprising the steps of: (a)providing DNA-molecules, comprising the coding regions for the at leastone surface to be mutated of the first chain or second chain, in (a)joint or separate mutagenesis-vector system(s), (b) mutagenesis of theDNA-molecules in a manner known as such, wherein the nucleic acidsequence encoding for the at least one surface is modified compared tothe initial sequence in such a manner that in the at least one surfaceof the first chain or the at least one surface of the second chain, asterically projecting group is introduced, and in the corresponding atleast one interacting surface of the second chain or the first chain, asterically recessed group is introduced, whereby individual mutatedfragments are produced, c) translation of at least two of the singlemutated fragments from step b), such that the pairing of theheterodimeric specific first-chain/second-chain TCR being mutated atleast one surface is selectively promoted, and d) presentation of theheterodimeric first-chain/second-chain TCR by a T-cell.
 21. The mutatedTCR, according to claim 20, which is mutated MDM2(81-88)-specific TCR(Seq ID No. 2 and Seq ID No. 5).
 22. An isolated nucleic acid,comprising a sequence coding for a mutated alpha- or beta-chain of a TCRproduced according to a method for producing a heterodimeric specificwild type- or chimeric T-cell receptor (TCR), containing a first chainand a second chain that interact one with another at at least onesurface, wherein the at least one surface is subjected to a rationalmutagenesis, such that the at least one surface of the first chain orthe surface of the second chain comprises a sterically projecting group,which interacts with a sterically recessed group on the at least onesurface of the corresponding first chain or second chain, comprising thesteps of: (a) providing DNA-molecules, comprising the coding regions forthe at least one surface to be mutated of the first chain or secondchain, in (a) joint or separate mutagenesis-vector system(s), (b)mutagenesis of the DNA-molecules in a manner known as such, wherein thenucleic acid sequence encoding for the at least one surface is modifiedcompared to the initial sequence in such a manner that in the at leastone surface of the first chain or the at least one surface of the secondchain, a sterically projecting group is introduced, and in thecorresponding at least one interacting surface of the second chain orthe first chain, a sterically recessed group is introduced, wherebyindividual mutated fragments are produced, c) translation of at leasttwo of the single mutated fragments from step b), such that the pairingof the heterodimeric specific first-chain/second-chain TCR being mutatedat least one surface is selectively promoted, and d) presentation of theheterodimeric first-chain/second-chain TCR by a T-cell.
 23. Acomposition selected from the group consisting of: a) a DNA- orRNA-vector molecule, comprising at least one nucleic acid according toclaim 22, and that can be expressed in cells; b) a host cell containinga DNA- or RNA-vector molecule comprising at least one nucleic acidaccording to claim 22; and c) a recombinant T-cell, expressing at leastone mutated alpha- or beta-chain of a TCR, produced according to amethod for producing a heterodimeric specific wild type- or chimericT-cell receptor (TCR), containing a first chain and a second chain thatinteract one with another at at least one surface, wherein the at leastone surface is subjected to a rational mutagenesis, such that the atleast one surface of the first chain or the surface of the second chaincomprises a sterically projecting group, which interacts with asterically recessed group on the at least one surface of thecorresponding first chain or second chain, comprising the steps of: (i)providing DNA-molecules, comprising the coding regions for the at leastone surface to be mutated of the first chain or second chain, in (a)joint or separate mutagenesis-vector system(s), (ii) mutagenesis of theDNA-molecules in a manner known as such, wherein the nucleic acidsequence encoding for the at least one surface is modified compared tothe initial sequence in such a manner that in the at least one surfaceof the first chain or the at least one surface of the second chain, asterically projecting group is introduced, and in the corresponding atleast one interacting surface of the second chain or the first chain, asterically recessed group is introduced, whereby individual mutatedfragments are produced, (iii) translation of at least two of the singlemutated fragments from step b), such that the pairing of theheterodimeric specific first-chain/second-chain TCR being mutated atleast one surface is selectively promoted, and (iv) presentation of theheterodimeric first-chain/second-chain TCR by a T-cell. 24-26.(canceled)
 27. A method for treatment of a cancerous disease, comprisingproviding to a patient in need thereof at least one of the following.(a) a mutated alpha- or beta-chain of a TCR, produced according to amethod for producing a heterodimeric specific wild type- or chimericT-cell receptor (TCR), containing a first chain and a second chain thatinteract one with another at at least one surface, wherein the at leastone surface is subjected to a rational mutagenesis, such that the atleast one surface of the first chain or the surface of the second chaincomprises a sterically projecting group, which interacts with asterically recessed group on the at least one surface of thecorresponding first chain or second chain, comprising the steps of: (i)providing DNA-molecules, comprising the coding regions for the at leastone surface to be mutated of the first chain or second chain, in (a)joint or separate mutagenesis-vector system(s), (ii) mutagenesis of theDNA-molecules in a manner known as such, wherein the nucleic acidsequence encoding for the at least one surface is modified compared tothe initial sequence in such a manner that in the at least one surfaceof the first chain or the at least one surface of the second chain, asterically projecting group is introduced, and in the corresponding atleast one interacting surface of the second chain or the first chain, asterically recessed group is introduced, whereby individual mutatedfragments are produced, (iii) translation of at least two of the singlemutated fragments from step b), such that the pairing of theheterodimeric specific first-chain/second-chain TCR being mutated atleast one surface is selectively promoted, and (iv) presentation of theheterodimeric first-chain/second-chain TCR by a T-cell; or (b) arecombinant T-cell, expressing at least one mutated alpha- or beta-chainof a TCR, produced according to a method for producing a heterodimericspecific wild type- or chimeric T-cell receptor (TCR), containing afirst chain and a second chain that interact one with another at leastone surface, wherein the at least one surface is subjected to a rationalmutagenesis, such that the at least one surface of the first chain orthe surface of the second chain comprises a sterically projecting group,which interacts with a sterically recessed group on the at least onesurface of the corresponding first chain or second chain, comprising thesteps of: (i) providing DNA-molecules, comprising the coding regions forthe at least one surface to be mutated of the first chain or secondchain, in (a) joint or separate mutagenesis-vector system(s), (ii)mutagenesis of the DNA-molecules in a manner known as such, wherein thenucleic acid sequence encoding for the at least one surface is modifiedcompared to the initial sequence in such a manner that in the at leastone surface of the first chain or the at least one surface of the secondchain, a sterically projecting group is introduced, and in thecorresponding at least one interacting surface of the second chain orthe first chain, a sterically recessed group is introduced, wherebyindividual mutated fragments are produced, and (iii) translation of atleast two of the single mutated fragments from step ii), such that thepairing of the heterodimeric specific first-chain/second-chain TCR beingmutated at least one surface is selectively promoted; and (iv)presentation of the heterodimeric first-chain/second-chain TCR by aT-cell.
 28. The method according to claim 27, wherein a cancerousdisease is treated that is in connection with a modified expression ofMDM2, p53, Her-2/neu, Ras, tyrosinase, MART, Gp100, MAGE, BAGE, MUC-1,CD45, CD19 or PRDI-BF1.